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Abstract

Background

The small ruminant parasite Haemonchus contortus is the most widely used parasitic nematode in drug discovery, vaccine development
and anthelmintic resistance research. Its remarkable propensity to develop resistance
threatens the viability of the sheep industry in many regions of the world and provides
a cautionary example of the effect of mass drug administration to control parasitic
nematodes. Its phylogenetic position makes it particularly well placed for comparison
with the free-living nematode Caenorhabditis elegans and the most economically important parasites of livestock and humans.

Results

Here we report the detailed analysis of a draft genome assembly and extensive transcriptomic
dataset for H. contortus. This represents the first genome to be published for a strongylid nematode and the
most extensive transcriptomic dataset for any parasitic nematode reported to date.
We show a general pattern of conservation of genome structure and gene content between
H. contortus and C. elegans, but also a dramatic expansion of important parasite gene families. We identify genes
involved in parasite-specific pathways such as blood feeding, neurological function,
and drug metabolism. In particular, we describe complete gene repertoires for known
drug target families, providing the most comprehensive understanding yet of the action
of several important anthelmintics. Also, we identify a set of genes enriched in the
parasitic stages of the lifecycle and the parasite gut that provide a rich source
of vaccine and drug target candidates.

Conclusions

The H. contortus genome and transcriptome provide an essential platform for postgenomic research in
this and other important strongylid parasites.

Background

Resistance to broad spectrum anthelmintic drugs is now widespread in parasites of
domestic livestock [1,2] and there are increasing concerns about the sustainability of human parasite control
programs using mass administration of the same drugs [3]. Consequently, there is an urgent need to understand the genetic mechanisms underlying
anthelmintic resistance and to discover new methods of chemical and non-chemical control.
However, the genomic and genetic resources required to underpin research in parasitic
nematodes are lacking [4-6]. The free-living nematode Caenorhabditis elegans is a powerful model system, but it has clear limitations for the study of parasitic
species [7]. Although the need to develop workable parasitic nematode model systems is widely
recognized, most human helminth species are not amenable to experimental study. In
contrast, Haemonchus contortus, a gastrointestinal parasitic nematode of small ruminants, has a successful track
record in anthelmintic resistance [7,8], drug discovery [9,10] and vaccine [11-13] research. It is amongst the most experimentally tractable parasites for a number
of reasons: adult females are relatively large and produce thousands of eggs per day,
allowing the production of large amounts of biological and genetic material, the infective
larvae (L3) can be viably cryopreserved, and in vivo studies, including genetic crosses, can be undertaken in the natural host [8]. Its phylogenetic position within the most closely related group of parasites to
C. elegans facilitates comparative genomics and heterologous gene expression, allowing functional
studies to be performed on H. contortus genes and regulatory elements [4,14]. As this parasite is a strongylid nematode, research on it is of particular relevance
to the most economically significant parasites of grazing ruminants and to the human
hookworms [15]. H. contortus itself causes major economic loss in small ruminants worldwide; it is highly pathogenic
and unsurpassed in its ability to develop resistance to every anthelmintic used in
its control. Notably, these include a number of core drugs used for mass drug administration
programs in humans [3]. Anthelmintic resistance in H. contortus and related strongylids now threatens the viability of the sheep industry throughout
the world [2]. This represents both a warning and a useful model for the consequences of the widespread
intensive use of anthelmintics that are now being used to control human parasites
in the developing world [3].

H. contortus has a direct and rapid lifecycle (Figure S1 in Additional file 1); adults reside and mate in the abomasum of the ruminant host, then females produce
eggs to be excreted in the feces. The eggs embryonate, develop and hatch as first
stage larvae (L1), develop and molt to become second stage larvae (L2), then molt
again to become third stage larvae (L3) in the feces. The L3 migrate onto pasture
to be ingested by the grazing ruminant host. The L3 shed the retained L2 cuticle (exsheath)
in the rumen, travel to the abomasum and develop through to the fourth larval stage
(L4) then adults in two to three weeks [16]. As voracious blood-feeders, H. contortus L4 and adults can cause severe hemorrhagic gastritis, hypoproteinemia, anemia and
edema, with acute infections resulting in death of the animal host. One adult female
can produce up to 10,000 eggs per day [17] and a single animal can harbor thousands of worms. This extremely high fecundity,
under conducive host and environmental conditions, can give rise to population explosions
and devastating disease outbreaks. Development occurs most rapidly in warm humid conditions,
but the L4 stage can undergo arrested development within the host to survive adverse
conditions such as prolonged drought or cold winters [18]. This feature of facultative arrested development, aided by movement of domestic
livestock and climate change, has ensured the worldwide distribution and success of
this parasite even though its evolutionary origins were in sub-Saharan Africa [19].

One striking feature of H. contortus, in common with related nematode species, is the extremely high level of genetic
diversity that has been reported in both laboratory and field populations; this is
thought to be predominantly a function of its large effective population size [20-22]. Genetic variation may underlie both the parasite's remarkable ability to adapt to
different climatic regions and host species [23] and its alarming propensity to develop drug resistance. This high level of genetic
polymorphism has also provided a major challenge to genome assembly, necessitating
the production of an inbred line from which to prepare DNA template.

In this paper, we describe the assembly and annotation of the draft genome of MHco3(ISE).N1,
a genetically well characterized inbred H. contortus strain that is susceptible to all major anthelmintic drugs. The most extensive comparative
transcriptomic sequencing and analysis yet described for a parasitic nematode was
undertaken on representative life-stages and the nematode gut to explore different
aspects of nematode biology, evolution and parasitism. This draft genome provides
a platform for future research in anthelmintic resistance, drug discovery and vaccine
development using H. contortus, currently the most important experimental model for the strongylid nematode group.

Results and discussion

Genome structure and content

We assembled a draft sequence of the H. contortus genome based on data from a mixture of sequencing technologies (Material and methods;
Additional results and methods in Additional file 1). Our final draft assembly consists of 67,687 contigs linked into 26,044 scaffolds
of total length 370 Mb, including 23.8 Mb of inferred gaps between contigs, with an
average and N50 scaffold length of 14,206 and 83,287 bp, respectively (Table S1 in
Additional file 1). This is a significantly larger genome size than the approximately 60 Mb predicted
with flow cytometry [24] but it is consistent with a prediction of approximately 200 to 300 Mb inferred from
a pilot annotation of two large contiguous regions of the genome [14], making this the largest nematode genome sequenced to date (Table S1 in Additional
file 1). The overall base composition of all sequence contigs was close to neutral at a
mean 41.3% GC, slightly higher than that for other clade V nematodes. Approximately
93% of conserved eukaryotic genes can be identified in the assembly, suggesting that
our draft assembly represents at least that fraction of the H. contortus genome [25], as many of these models are incomplete or split between contigs (Table S2 in Additional
file 1). Despite this, our current assembly is of similar quality to some other published
draft nematode genome sequences [26-30] (Table S1 in Additional file 1).

We used extensive transcriptomic evidence from across the H. contortus lifecycle (Materials and methods; Table S3 in Additional file 1) to guide de novo gene model curation for protein-coding genes. We predict a similar number of protein-coding
genes to the C. elegans genome (21,799 versus 20,532), but significantly lower gene density of 59 genes per
Mb, with only 7% of the genome being protein coding, compared to 200 genes per Mb
and a protein coding content of approximately 30% in the C. elegans genome (Figure 1). The average coding sequence length is similar in H. contortus and C. elegans (1,127 bp in H. contortus compared to 1,371 bp in C. elegans) but the average gene length is more than double in the parasite (6,564 bp compared
to 3,010 bp in C. elegans). A closer look at a subset of 2,822 H. contortus and C. elegans one-to-one orthologs shows the discrepancy is explained by an expansion in both the
number and length of introns in H. contortus (average of 10 introns per gene, average size 633 bp; relative to 6 introns per gene,
average size 340 bp in C. elegans). An expansion in intronic sequence has also been reported in the closely related
necromenic nematode Pristionchus pacificus but to a less dramatic extent (average of nine introns per gene, average size 309
bp) [26]. Over 80% of H. contortus and C. elegans one-to-one orthologs present on the same scaffold occur on the same C. elegans chromosome (Table S4 in Additional file 1), suggesting widespread conservation of synteny between the two species (Figure 2). However, gene order is generally poorly conserved, which is consistent with comparisons
between C. elegans and related nematodes [27,31], but regions of conserved microsynteny are apparent and may prove useful in supporting
orthology of divergent genes.

Figure 1.Evolution of genome content in clade V nematodes. A maximum-likelihood phylogeny based on concatenated alignment of single-copy genes.
Values on edges represent the inferred numbers of births (+) and deaths (-) of gene
families along that edge. Note that our approach cannot distinguish gene family losses
from gains on the basal branches of this tree, so, for example, the value of 1,263
gene family gains on the basal branch of clade V will include gene families lost on
the branch leading to the clade IV species. The first column of pie charts represents
the gene family composition of each genome - the area of the circle is proportional
to the predicted proteome size, and wedges represent the numbers of proteins predicted
to be either singletons (that is, not members of any gene family), members of gene
families common to all nine genomes, members of gene families present only in a single
genome, and members of all other gene families. The second column of pie charts represents
the genomic composition of the species with published genome sequences. All ingroup
nodes had 100% bootstrap support.

Figure 2.One-to-one orthologs connecting H. contortus scaffolds and C. elegans chromosomes. (a) All 130 H. contortus scaffolds with at least five one-to-one orthologs between the two species, with scaffolds
ordered to maximize colinearity with the C. elegans genome. (b) The relative positions of one-to-one orthologs between the two largest H. contortus scaffolds and C. elegans chromosomes, demonstrating the lack of conservation of gene order within syntenic
blocks.

Reflecting this relatively expanded genome, approximately 29% of the draft genome
assembly consists of repetitive sequence (compared to approximately 16% in C. elegans), including 2,434 copies of the characteristic HcRep element previously reported
from a number of trichostrongylid species [14,32-34]. The repetitive sequence also includes representatives of many known repeat families
in other nematodes, with approximately 6% of the genome derived from LINEs, 2.5% from
long terminal repeat retrotransposons and 5% from DNA transposons, including TTAA-specific,
Mariner-like and MuDR-superfamily elements, together with evidence of elements related
to Tc1 and Tc4 of C. elegans. Despite belonging to similar families, H. contortus repeats represent independent expansions to those in other clade V nematodes, as repeat
libraries from H. contortus show little similarity to genome sequence from other species, and vice versa. Our
transcriptomic data suggest that active transposition is occurring, with evidence
of expression for 4 out of 26 gene models annotated with transposase domains and 49
out of 482 reverse transcriptase domain-containing proteins. Expressed proteins appear
to belong to a range of DNA transposon types, and to Gypsy-related and LINE retro-elements.

In C. elegans, around 17% of genes are in operons [35] - tightly linked clusters of two to eight genes, which are co-transcribed from the
same promoter. The resulting polycistronic pre-mRNAs are resolved by trans-splicing with spliced leader (SL) SL1 and SL2 sequences. Most frequently, SL1 is
trans-spliced to the first gene in an operon and downstream genes are SL2 trans-spliced. The structure (gene complement, order and orientation) of around 23% of
C. elegans operons is conserved in the H. contortus genome. The structure of a further 10% of C. elegans operons appear to be partially conserved, where at least two orthologs are present
on the same scaffold in the expected order and orientation, but one or more genes
are in a different order, inverted or absent. Functional constraints are thought to
conserve the intergenic distance in C. elegans operons to approximately 100 bp but genes in H. contortus operons are further apart: the average intergenic distance of genes with a conserved
operon structure is 992 bp (median 621 bp, largest 8,329 bp), and the operon encoding
ion channel subunits Hco-deg-2H and Hco-deg-3H has an intergenic distance of 2,342
bp [36].

Overall, SL1 trans-splicing was detected in 6,306 H. contortus genes and SL2 trans-splicing was detected in 578 genes. Of these, 318 trans-spliced genes were in the putative conserved operons identified above (Additional
methods in Additional file 1). All 126 first genes in operons were trans-spliced to SL1 (SL2 trans-splicing was detected in five putative first genes, but examination of their loci
suggests they are downstream genes in new operons in this species); 119 downstream
genes were trans-spliced to SL1 and 73 were trans-spliced to SL2. If SL2 trans-splicing is the definitive criterion in identifying operons, the relatively low level
of SL2 trans-splicing to downstream genes suggests that either operon function is
less frequently conserved than operon structure in H. contortus or that undetected, divergent SL2 sequences are present. However, the relatively high
frequency of SL1 trans-splicing in genes that are also SL2 trans-spliced (approximately 77%; 56 of 73 in conserved operons, 445 of 578 in all genes
identified) suggests SL1 trans-splicing of downstream genes may also be relatively common in this species.

We employed two complementary approaches to global comparison of the H. contortus gene set, using the Inparanoid algorithm to look in detail at orthologs with C. elegans and P. pacificus, and OrthoMCL for a wider view of gene family evolution with other clade V nematodes.
Of 5,937 orthology groups between C. elegans and H. contortus, 5,012 are one-to-one orthologs, while an additional 899 orthologs could be identified
in H. contortus and P. pacificus but not C. elegans, suggesting they have been lost in the C. elegans lineage (Table S5 in Additional file 1). A number of orthology groups are significantly expanded in H. contortus, including a family of 180 Haemonchus paralogs to a single C. elegans gene that lacks any functional annotation (Table S6 in Additional file 1). Other expanded groups include genes with likely roles in parasitism, such as cysteine-rich
secreted proteins, together with a set of helicase domains that include some with
predicted signal peptides. Global analysis of the evolution of entire gene families
(clusters of similar genes) across the clade V nematodes confirms this pattern of
significant diversity within the clade (Figure 1), and allowed us to identify H. contortus genes lacking clear orthologs in other clade IV or V nematodes (Figure S2 and Table
S8 in Additional file 1). This shows that the Haemonchus-specific proteome is enriched in genes encoding polypeptides involved in proteolysis,
neurotransmission and carbohydrate metabolism, and in secreted proteins and those
expressed in the cuticle. While some of these genes are explored in detail in the
more focused analyses below, others may represent novel candidate genes involved in
the host-parasite interface.

Gene expression in parasite life-stages and gut

As H. contortus progresses through its lifecycle, it must adapt to different environments with differing
food sources and energy requirements (Figure S1 in Additional file 1) and this is reflected in differential gene expression. RNA-seq was used to analyze
gene expression in six parasite life-stages and the adult female gut. Samples were
made in triplicate from independent batches of parasite material for every stage,
allowing statistically robust comparison of relative gene expression between the stages
(Materials and methods; Table S3 in Additional file 1). We found significant expression for 17,483 genes in total from the 6 life-stages
studied; with between 13,962 (in L3) and 15,569 (in adult males) genes expressed in
each stage. A total of 11,295 genes were significantly up- or down-regulated through
the lifecycle (Figure 3 and Materials and methods) and we used annotation with Gene Ontology (GO) terms to
investigate their broad functions. Metabolic enzyme expression throughout the parasite
lifecycle was analyzed in more detail and will be discussed separately.

Figure 3.Differential gene expression across the H. contortus lifecycle. (a) Numbers of genes significantly up-regulated (red) and down-regulated (blue) between
life-stages. (b) The expression profiles of genes determined to be differentially expressed between
any pair of stages are shown in clusters ordered by the stage at which they are most
highly expressed. The innermost circle shows normalized expression levels during the
egg stage with further concentric circles showing expression in the L1, L3, L4 stages,
followed by male worms, female worms and then female gut samples. Mean expression
values were taken over three replicates and are shown on a log2 scale.

Genes up-regulated in the development from egg to the L1 stage include those associated
with muscle development and motor activity, while up-regulated in the egg are genes
associated with oxidoreductase activity, apoptosis, body morphogenesis and development,
larval and embryo development, as well as DNA replication and chromosome organization.
This pattern is consistent with the progression from a developing embryonated egg
to a motile and actively feeding larval stage.

In the transition to the L3 stage, a decrease in the expression of genes associated
with the myosin complex and motor activity and various metabolic processes are consistent
with the nematode entering a quiescent state. Among the up-regulated genes there is
an association with oxygen transport and heme binding. Oxidoreductase enzymes are
over-represented and may reflect the increased need to detoxify a build up of endogenous
waste, consistent with previous studies showing higher cytochrome P450 (CYP) activity
in the H. contortus L3 than in L1 or adult stages [37]. CYPs have also been shown to be up-regulated in response to reduced food intake
[38]. This is consistent with the significant increase in gluconeogenesis from the L1
to L3 - which is also increased in the C. elegans dauer [39] - and the up-regulation of acetyl-CoA metabolic process, likely to reflect metabolism
of fat stores in the non-feeding L3. Genes associated with binding of cobalamin (vitamin
B12) are also up-regulated. Cobalamin has been shown to be strongly concentrated and
stored in the infective L3 of other gastrointestinal nematodes [40] and a ready supply may be required for rapid larval development after ingestion by
the host.

The L4 is the first blood-feeding stage of H. contortus. The transition from the quiescent L3 stage to the motile L4 stage is reflected in
significant up-regulation of many genes, including genes associated with motor activity,
the myosin complex and locomotion as well as various metabolic processes. The binding
of oxygen, lipids and sugars, possibly associated with active feeding, is also up-regulated,
as are changes in the expression of genes linked to response to oxidative stress that
may reflect the reactivation of the parasite from its dormant stage. A significant
increase in the expression of genes associated with collagen and cuticle development
and body morphogenesis, consistent with parasite growth, is also observed. Interestingly,
heme-binding genes were both up- and down-regulated, perhaps reflecting an increase
in heme load from blood feeding and a decrease in CYP activity.

Transition from the L4 to the adult stages is characterized by several changes. In
the transition to the female stage, 1,658 genes are up-regulated and correlate with
gender-specific development as well as embryogenesis (adult females contain oocytes
and eggs at various developmental stages), such as genitalia development, embryo development,
oogenesis, ovulation, germ-line cell cycle switching, meiosis regulation and regulation
of vulval development. A significant increase in DNA replication processes is also
apparent. Between the L4 and adult male stage, lower expression in the adult male
of genes linked with body morphogenesis, molting, collagen and cuticle development,
oxidoreductase activity, heme binding and response to oxidative stress were observed
among the various alterations in expression. Increased expression of genes annotated
with the GO term 'structure molecule activity' is due to up-regulation of a number
of major sperm protein genes. These sex-specific gene expression patterns were confirmed
by comparison between adult female and male parasites, and it is clear that many genes
are highly enriched in male worms, being expressed only at low levels in other stages.
The large set of genes highly expressed in both eggs and adult females were again
apparent.

Finally, we investigated gene expression in the H. contortus intestine, the major organ of digestion and detoxification in the nematode, by comparing
the female gut sample with the whole female worm. Consistent with data from H. contortus gut EST libraries [41], increased expression of genes with protein kinase, cysteine-type peptidase and cysteine-type
peptidase inhibitor activities predominated. Genes associated with sugar and cobalamin
binding were significantly up-regulated, as were genes associated with transport of
cations, anions and oligopeptides. Oxidoreductase activity was also increased, consistent
with the expression pattern of detoxification genes in C. elegans [42].

Metabolic pathways and chokepoint analysis

Comparisons between H. contortus and the free-living nematodes revealed 22 enzyme classifications (ECs) that were restricted
to the parasite (Table S9 in Additional file 1). While more detailed analysis is required, metabolism of amino acids and carbohydrates
clearly differ between these two groups. For example, lysine 6-aminotransferase (EC
2.6.1.36) catalyzes lysine to glutamate, which can be further converted to α-ketoglutarate,
an intermediate of the tricarboxylic acid cycle. Lysine 6-aminotransferase was previously
considered restricted to prokaryotes; thus, its activity in H. contortus needs to be confirmed.

A summary of up- and down-regulated metabolic enzymes across all life-stages is shown
in Table S10 in Additional file 1. The transition through eggs, L1, L3 and L4 showed a striking pattern: from L1 to
L3, most enzyme classifications were down-regulated, including those involved in carbohydrate,
lipid and energy metabolism, but many of these were up-regulated again in the transition
to L4. This is consistent with the L3 being a stage in which development is arrested,
analogous to the dauer larva in C. elegans. Further support for this comparison is the up-regulation of two enzymes that independently
convert isocitrate to 2-oxo-glutarate, while most other parts of the tricarboxylic
acid cycle are down-regulated. Furthermore, 2-oxo-glutarate is an entry metabolite
into the ascorbate and aldarate metabolic pathway, which is implicated in increased
lifespan in Drosophila [43]. The L4-to-male transition shows a decrease in lipid metabolism coupled with an increase
in amino acid metabolism.

Metabolic chokepoints - reactions that uniquely consume or produce a metabolite -
are enzymes that seem likely to be essential to the parasite and so may be potential
targets for future drug development. Analysis of the H. contortus network revealed 362 chokepoint reactions, five of which passed additional criteria
of lacking isoenzymes and being divergent from known human enzymes [44] (Table S11 in Additional file 1). Comparison of these against the Therapeutic Target Database revealed that two are
known potential targets for anthelmintic drugs: trehalose-6-phosphatase (EC 3.1.3.12)
is a member of the sucrose metabolism pathway, and is currently being researched as
a potential drug target in the filarial nematode B. malayi [45], while dTDP-4-dehydrorhamnose 3,5-epimerase (EC 5.1.3.13) is currently of interest
in Mycobacterium tuberculosis [46]. This validation of the approach justifies further analysis of the other three chokepoint
enzymes identified.

Neuromuscular drug targets

In nematodes, the pentameric ligand-gated ion-channel (pLGIC) family is particularly
numerous, with 64 members identified in H. contortus via homology with Caenorhabditis spp., P. pacificus and RNA-seq data (Figure 4). They are of great importance in parasitic nematodes because they are targets of
the majority of the currently available anthelmintic drugs (as summarized in Table
S12 in Additional file 1; the β-tubulin targets of the benzimidazole group have been described previously
[47]).

Figure 4.Maximum-likelihood phylogenies of H. contortus and C. elegans ligand-gated ion channel families. Figure shows unrooted trees for (a) cation channel and (b) anion channel genes from the two nematode species, with different colors representing
each major clade of channels. Within clades, C. elegans genes are colored a lighter shade than H. contortus genes. Both trees show a general pattern of conserved one-to-one orthology, and colored
bars indicate where this pattern is broken - by a duplication in H. contortus (with four copies of unc-29), and by loss of some genes - there are four H. contortus genes (acr-26, acr-27, glc-5, glc-6) without orthologs in C. elegans, and seven in C. elegans missing from H. contortus (acr-13/lev-8, acr-18, acr-23, glc-1, lev-8, lgc-48, lgc-54). All of these genes are shown in large bold type. A truncated ortholog of C. elegans acr-9 is present in H. contortus but is omitted from this phylogeny. Values near internal nodes are Shimodaira-Hasegawa
test support values - with support values of 1.0 on all other nodes omitted for clarity.

The pLGICs regulate the flow of anions, typically chloride ions, or cations, including
sodium and calcium, in response to an extracellular signal in the form of an activating
ligand or change in pH. They are fundamental to synaptic transmission; interference
with their normal function results in paralysis and death. Drugs that activate the
anionic channels, such as the macrocyclic lactone ivermectin (IVM), typically inhibit
neuronal transmission and muscle contraction. Those that activate the cationic channels,
such as levamisole (LEV) and monepantal, stimulate neuronal transmission and typically
induce muscle contraction. Here we present the most complete picture of these channels
to date and show that, as expected, this parasite is very similar to C. elegans. This supports the use of the free-living worm as a functional model for the parasite
nervous system. There are, however, some important differences, most significantly
in glutamate signaling, which is sensitive to the macrocyclic lactones, and acetylcholine
signaling, which is sensitive to LEV, as well as characteristic loss of some receptor
genes associated with biogenic amine signaling.

The macrocyclic lactones, which include IVM and moxidectin, act at several different
glutamate-gated chloride channels. Some of these are found in both C. elegans and H. contortus, but it is noteworthy that two H. contortus subunit genes, glc-5 and glc-6, encode glutamate-sensitive channels that are absent from C. elegans. It is likely these were lost from the rhabtidid lineage as homologous sequences
can be detected in the genome of the close relative P. pacificus. Both of these subunits are targets for the macrocyclic lactones [48,49] and changes in either their sequence or expression have been associated with drug
resistance in veterinary parasites [50]. The archetypal target of IVM, Cel-glc-1, appears to be a duplication of avr-15, specific to C. elegans. These differences may explain the difficulties encountered in understanding IVM's
mode of action and resistance in parasitic nematodes by studying the model organism
[51]. Most of the other anionic channel subunits in H. contortus have direct orthologs in C. elegans (Figure 4a). The only notable absences are lgc-48 and lgc-54. lgc-48 encodes an as yet uncharacterized member of the acc-1 acetylcholine gated chloride channels [52] that appears to have been lost from H. contortus, and lgc-54 encodes an uncharacterized member of the C. elegans biogenic amine-gated chloride channel family that may not be present in H. contortus [53].

The acetylcholine gated receptor cation channels are of particular interest as they
are the targets of several anthelmintic drugs already in use (LEV, monepantel (MPTL)
and derquantel) and are considered to be one of the most promising gene families for
the identification of new drug targets. Comparison of the H. contortus gene family with those found in C. elegans is shown in Figure 4b. There is a one-to-one correspondence in H. contortus for subunits of the acr-16 clade [54], named for the homomeric nicotine-sensitive channel in C. elegans, and acr-16 expression is much higher in adult males than in other life-stages, as previously
described for C. elegans [55]. The anthelmintic LEV targets receptors composed of α subunits of the unc-38 clade and non-α subunits from the unc-29 clade. In C. elegans three different alpha and two different non-alpha subunits combine to form a channel
that responds to acetylcholine, strongly to LEV and only weakly to nicotine [56]. The α-type acr-13 (lev-8) gene, required in C. elegans for the LEV receptor, appears to have been lost in H. contortus as an ortholog of this gene is detectable in P. pacificus. The non-alpha gene lev-1 is present, but the signal peptide appears to have been lost [57]. A LEV receptor in H. contortus can be reconstituted without either of these, requiring only UNC-38, UNC-63 and UNC-29
as in C. elegans [57] and replacing ACR-13 with ACR-8. Most strikingly, the non-α subunit gene unc-29 in C. elegans corresponds to four paralogous copies in H. contortus [57]. A LEV-sensitive channel has been reconstituted containing the Hco-UNC-29.1 subunit
[58]. The degree to which the other copies may have diverged in function is currently
under investigation. The deg-3 clade [54] encodes subunits that form channels involved in chemotaxis, and is of particular
interest as channels encoded by des-2/deg-3 and acr-23 in C. elegans are targeted by the relatively new anthelmintic MPTL, with acr-23 as the principal target of MPTL action in vivo [9]. The single subunit gene mptl-1 in H. contortus corresponds to the acr-23/acr-20 pair in C. elegans. Splice site mutations in the mptl-1 gene are associated with resistance to MPTL [36], which would suggest that acr-23 is functionally equivalent to mptl-1.

H. contortus possesses two acetylcholine gated receptor genes that are not present in C. elegans, acr-26 and acr-27, and these two genes seem to form a distinct clade, which suggests that they are
likely to form receptors with a distinct pharmacology. Orthologs of acr-26 are found in many species of parasitic nematode and therefore they might make interesting
targets for the development of novel cholinergic anthelmintics.

In summary, the majority of the pLGIC subunit genes in C. elegans have direct orthologs in H. contortus, although there is more variation in the repertoire of orphan family pLGICs (Additional
results in Additional file 1). This supports the use of C. elegans as a model to study the neuromusculature of H. contortus in drug screens, but there are important differences in specific anthelminthic targets:
for each of the anthelmintics IVM, LEV and MPTL, the drug targets in H. contortus are functionally different to the C. elegans model.

Drug metabolism and efflux

Parasitic nematodes are armed with a large repertoire of inducible metabolizing enzymes
and transporters to protect against environmental toxins. The nematode detoxification
pathway can be divided into three main phases: modification, conjugation and excretion
[59] involving the cytochrome P450s (CYPs) and the short-chain dehydrogenase/reductases
(SDRs) in phase 1, the UDP-glucoronosyl transferases (UGTs) and the glutathione S-transferases
(GSTs) in phase 2 and the ATP-binding cassette (ABC) transporters in phase 3. Little
is known about the impact of the parasite detoxification system on anthelminthic efficacy
or resistance [60], but a better understanding of these pathways will allow their role in anthelmintic
resistance to be assessed and should also be informative in the design of new drugs
and synergistic agents [61]. We have annotated a large number of modification and conjugation genes in the current
assembly, identifying a total of 42 CYPs, 44 short-chain dehydrogenase/reductases,
34 UDP-glucoronosyl transferases and 28 glutathione S-transferases (Figure S5 in Additional
file 1), but here we focus on the excretion transporters implicated in drug resistance.

Members of the ABC transporter family hydrolyze ATP and couple the energy released
with the active transport of a wide range of compounds, including small organic molecules,
lipids, proteins and metal ions. They are an essential component of many biological
processes and are fundamental to the barrier between a nematode and its environment.
These transporters consist of a basic structure of six transmembrane domains with
an associated intracellular ATP binding motif. The functioning transporter complex
requires two of these protein halves, but some members of this family are fusion proteins
with 12 transmembrane domains and two ATP binding motifs. We find numerous differences
in ABC transporter genes between C. elegans and H. contortus (Additional results and Figure S4 in Additional file 1), with a reduced complement of haf-transporters, including, for example, the loss
of haf-6, which is essential for efficient RNA interference in C. elegans [62], a significant expansion of ced-7, which has an unknown function in amphid and phasmid sensory cells, and an extensive
change in the repertoire of multidrug resistance protein genes. The P-glycoprotein
(pgp) transporters are of particular interest as they have been implicated in resistance
of H. contortus to IVM and other anthelmintics [49,63], and this family has undergone significant change compared to the free living C. elegans. In total, 10 P-glycoprotein genes have been identified in the H. contortus genome and knowledge of this full complement will now allow a more systematic analysis
of the role of P-glycoproteins in resistance to IVM and other anthelmintics. A cluster
of four C. elegans genes, pgp-5, 6, 7 and 8, are not found in H. contortus. Cel-pgp-3 and 4 as well as Cel-pgp-12, 13 and 14 represent gene duplication events corresponding to single genes in the parasite. Cel-pgp-9 corresponds to two paralogous copies in H. contortus, and in addition, two genes not present in C. elegans, related to pgp-3 and pgp-11, have been retained in H. contortus. These are named pgp-16 and pgp-17, respectively. Changes in the sequence or expression of pgp-1, pgp-2 and pgp-9 have been reported for IVM-resistant versus-susceptible isolates of H. contortus and in pgp-9 for resistant Telodorsagia circumcincta [50,64,65].

Protease vaccine candidates

H. contortus is a voracious blood-feeder, with even modest infections of 1,000 worms generating
losses of up to 50 ml of blood per day [66]. Cysteine, aspartic and metallo-proteases as well as aminopeptidases have been implicated
in important aspects of parasite function, including hemoglobin digestion and anticoagulant
activity, and these enzymes are also important vaccine candidates. Vaccination with
gut extracts enriched for these activities can confer up to 75% reduction in worm
burden and 90% reduction in egg output [67]. Protection is thought to result from the ingestion of host antibodies by the parasite
during blood-feeding, which bind to gut antigens and disrupt function. Female parasites
appear to be more affected than males, increasing the relative impact on egg output.
Development of a commercial vaccine, however, requires identification and expression
of specific proteases that, either singly or combined, induce protective immunity.
Comparative genomics and transcriptome data can aid target selection by identifying
potential functionally important proteases and those enriched in the gut of blood-feeding
L4 and adult stages, suggesting a role in blood-feeding as well as accessibility to
host antibodies.

Cathepsin B protease (cbl) genes are part of an ordered hemoglobin degradation pathway,
functioning after aspartic proteases (APRs) and upstream of metalloproteases (MEPs)
and aminopeptidases [68,69]. Cathepsin B diversity may therefore be key in generating an array of substrates
from ingested nutrients for efficient cleavage by downstream proteases and may be
involved in the high blood digestion capacity of H. contortus. Indeed, H. contortus has a higher copy number (63 genes) of cathepsin B protease genes than related free-living
nematodes, representing 80% of all cathepsin cysteine protease genes in the genome
(Figure 5a). This large expansion of the cathepsin B family has resulted in H. contortus showing a greater diversity of cbl genes than known in any other parasitic nematode.
Uniquely, members of each cbl type are arranged in tandem in the H. contortus genome (Figure 5b) and have the same genomic structure, suggesting that diversity has arisen through
recent gene duplication. Reflecting this, most CBLs encoded in the H. contortus genome form large monophyletic groups distinct from C. elegans [70], hookworm [71] or other strongylid CBLs, suggesting that duplication and divergence has occurred
separately following speciation. It is possible that gene amplification has occurred
as a mechanism of increasing overall CBL expression associated with the need for H. contortus to digest the huge quantities of host blood it takes in during feeding. The cbl genes
show increased expression in L4 and adult stages and many are significantly enriched
in gut tissue, identifying these as potentially important control targets. These include
both novel and previously identified cbl genes (AC-4, gcp-7 and hmcp-2) [72-74], while other cbl transcripts are significantly up-regulated in the L4 stage and in
adult male worms, but not in the gut, suggesting a role in development and reproduction
rather than feeding. The CBLs identified here contain a putative amino-terminal signal
peptide and several have been identified in adult worm excretory-secretory products
[75]. It has also been proposed that sequence variation of Hc-CBLs may confer antigenic
diversity [76], and presentation to the host immune system through secretion may therefore drive
the maintenance of the diversity of Hc-cbl genes. Three-dimensional modeling of the
CBL repertoire and epitope mapping will clarify this issue.

Figure 5.(a) Maximum-likelihood phylogeny of cathepsin B (cbl) genes in H. contortus and related nematodes. The H. contortus genome shows large, independent expansions of all three main classes of cbl genes:
AC (anticoagulant)-like, GCP-7-like and Hmcp-like. (b) Many of the genes in two of these expansions occur as tandem arrays of duplicates
on three scaffolds of the H. contortus genome. Two of these scaffolds are known to be linked, as eight of the genes occur
on a previously completed bacterial artificial chromosome (BAC) sequence.

Other cathepsin cysteine protease genes are not expanded - for example, a cpr-6-like single copy gene is highly conserved in a number of parasitic and free-living
nematodes, suggesting a house-keeping role, and there is no expansion found for cathepsin
L, F or Z genes in H. contortus. Furthermore, there is less expansion of genes encoding other vaccine candidates
- H-gal-GP composed predominantly of pepsinogen-like aspartic and metallo proteases
[77], and H11 aminopeptidases [78]. These are integral gut membrane proteins, considered hidden from the immune system
during natural infection [67], and lower diversity should be advantageous in vaccination studies.

Significant expansion is, however, seen in the cathepsin aspartic protease family
(Figure S6 in Additional file 1). Importantly, the genome data identify a novel, single-copy apr gene with 84% amino
acid identity to APR-1 of the dog hookworm Ancylostoma caninum and that is significantly enriched in gut tissue. Vaccination with recombinant Ac-APR-1
significantly reduced fecal egg counts, worm burdens and anemia [79], warranting investigation of the protective potential of the Haemonchus APR-1-related protease. Other novel Hc-apr genes group phylogenetically with C. elegans asp genes (Figure S6 in Additional file 1) and are increased in the environmental L1 stage, indicating a developmental role,
consistent with C. elegans data [80,81] and ruling these out as potential vaccine targets.

Conclusions

H. contortus is the one of the most intensively researched parasitic nematode species and is the
most established parasite species used in drug discovery, drug mode-of-action and
drug resistance research. It is the first strongylid nematode to have its genome sequenced
and the analysis presented here provides a first genomic insight into the biology
of a gastro-intestinal strongylid and blood feeding parasitic nematode. It confirms
the close relationship between H. contortus and C. elegans and provides a platform from which to apply the C. elegans biological knowledge to investigate the biology of this parasite for both basic and
applied research. It also highlights specific differences between the species that
will be important to inform researchers of those aspects of C. elegans biology that cannot be extrapolated. The genome sequence, gene models, transcriptome
datasets and bioinformatic analyses provide a wealth of information on potential new
drug and vaccine targets. They will also be an invaluable resource for the application
of post-genomic technologies to this and other related strongylid parasites.

Materials and methods

Parasite material production and quality assurance

The MHco3(ISE) line is a H. contortus strain, susceptible to all the major anthelmintic classes, that has been passaged
in the laboratory by serial infection for many years. It has been extensively phenotypically
and genetically characterized previously [82]. The inbred MHco3(ISE).N1 strain was derived by a genetically validated single pair
mating of an adult male and an adult female MHco3(ISE) worm using a method of direct
transplantation into the abomasum of a recipient parasite-free sheep (described in
[83]). The inbred MHco3(ISE).N1 strain was subsequently passaged by oral experimental
infection of parasite-free sheep and parasite material was harvested using standard
methods (see Additional methods in Additional file 1 for details). All parasite material used in this study was derived from the inbred
H. contortus MHco3(ISE).N1 strain except for the female gut transcriptomic data, which were generated
from the MHco3(ISE)strain. The MHco3(ISE) and MHco3(ISE).N1 strains were routinely
monitored for genetic integrity at each passage, as was all parasite material derived
from the strains, using a standard panel of microsatellite markers and a well established
'genetic fingerprinting' methodology [8,82]. Both the MHco3(ISE) and the MHco3(ISE).N1 are viably cryopreserved and available
on request.

Genome sequencing and assembly

We assembled a draft sequence of the H. contortus genome based on data from a mixture of sequencing technologies, including 21 × coverage
of paired-end and shotgun reads from a Roche454SLX platform and approximately 163
× coverage of long- and short-insert paired-end libraries from an Illumina HiSeq (coverage
based on a genome size of 370 Mb). Genomic and transcriptomic sequence data were generated
using largely standard molecular biology methods (Additional methods in Additional
file 1), except that whole-genome amplified material was used to generate sufficient material
for a large-insert Illumina library from a single male worm, using a modified protocol.
The genomic reads from each technology were initially assembled independently using
assembly algorithms most suited to the typical coverage and read length of each, and
scaffolds from each were then merged to form an initial set of scaffolds that were
then improved by automated gap filling, followed by breaking and re-scaffolding using
the paired-end data from both sequencing platforms.

Protein coding gene prediction and functional annotation

After quality control and end-trimming, the transcriptome reads were mapped against
the reference genome using TopHat software, v 2.0.6 (--mate-std-dev 50 -a 6 -i 10
-I 20000 --microexon-search --min-segment-intron 10 --max-segment-intron 50000) [84]. A reference dataset of 399 H. contortus protein encoding genes was manually curated from predictions of highly conserved genes
using CEGMA (version 2.4) [24] and RNA-seq mapping. Of those, 347 were used to train Augustus [85] and 52 were used to independently evaluate the accuracy of the predictions. Final
gene prediction (v2.0) was performed by Augustus using H. contortus specific parameters and RNA-seq, EST and polyA mappings as evidence hints generated
by TopHat2 and PASA [86], respectively. Gene prediction accuracy was computed at the level of nucleotides,
exons and complete genes on 52 manual curated gene models as described previously
[87] and shown in Table S7 in Additional file 1). Functional annotation information was obtained from the interpro databases using
interproscan v4.5 [88], GO [89] terms were annotated via interpro2GO and from the curated C. elegans annotation in Wormbase (release 235) [90] by assigning all GO terms shared by all C. elegans genes in a gene family to the H. contortus members of that family. Further functional insight was obtained by BLAST searches
for similar genes in the GenBank nr database, and putative signal peptides were identified
by SignalP [91]. To investigate H. contortus metabolism, a total of 828 ECs, covering 2,853 proteins, were assigned using KAAS
[92], DETECT [93] and EFICAz [94]. Of these, 563 ECs covering 1,246 proteins were assigned to a metabolic pathway;
the others are non-metabolic enzymes. Similar annotation efforts were carried out
on Caenorhabditis species and P. pacificus.

Gene expression and Gene Ontology analysis

The numbers of RNA-seq reads per gene model were counted using custom-made scripts
making use of BEDtools and a gff file of the genome annotation, and based on the TopHat
mapping described above. Analysis of gene expression was performed using the DESeq
(v 1.8.1) package for Bioconductor [95]. Read coverage was normalized to estimate the effective library sizes for each library
and negative binomial tests performed between pairs of sample triplicates, using dispersion
estimates from the default approach, to obtain P-values for differential expression of each gene adjusted for false discovery rate
using the Benjamini-Hochberg procedure for multi-testing [96]. Only genes with adjusted P-values ≤1e-5 were retained. GO terms enriched in the set of differentially expressed genes in each
comparison were identified using the 'weight01' algorithm of the TopGO (v 2.8.0) package
for Bioconductor [97]. Only GO terms with P < 0.01 were considered for more detailed analysis. Expression data were drawn using
Circos-0.62. All differentially expressed genes were clustered using MBCluster.seq
with 50 clusters, and clusters then ordered based on which stage had the highest mean
expression in that cluster.

Data access

All sequences described in this paper have been submitted to the GenBank database,
project ID PRJEB506. Sequence data are available at [98] and annotation has been submitted to GenBank and Wormbase. ENA accession numbers
for all genomic and RNA-seq reads are listed in Tables S3, S13 and S14 in Additional
file 1. The H. contortus genome assembly and functional annotation are available at [99].

Acknowledgements

The Haemonchus contortus genome project is funded by the Wellcome Trust through their core support of the Wellcome
Trust Sanger Institute (grant 098051) and a Wellcome Trust Project Grant to JSG (grant
067811). JSG also acknowledges funding from the Canadian Institutes of Health Research
(CIHR) 230927. JSG and JW acknowledge support from the NSERC-CREATE Training Program
in Host-Parasite Interaction at the University of Calgary (grant number 403888-2012).
RL is funded through the Scottish Government by the Strategic Partnership for Animal
Science Excellence (SPASE). SJ is funded through a BBSRC/KTN/Pfizer CASE studentship.
We thank Nathalie Smerdon, Andrew Knights and David Willey for technical support in
generating RNA-seq libraries; Coline Griffiths, Matthew Jones, Lesley Shirley, Naomi
Park and Mike Quail for Illumina genomic libraries; and Richard Rance for 454 genomic
libraries. Robert Davies and Quan Lin arranged for data release to the SRA. We thank
Doug Jasmer for the H. contortus gut dissection protocol.

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